In mammalian respiration, cytochrome c oxidase (CcO) is the final electron acceptor in the respiratory chain. CcO catalyzes the four-electron reduction of oxygen to water and pumps protons across the inner mitochondrial membrane to establish an electrochemical proton gradient that is used to synthesize ATP. As CcO plays a central role in mitochondrial energetics, and defects in CcO functionality are associated with important mitochondrial diseases, the enzyme has been under intense investigation. However, the mechanism of its major function, the coupling between the oxygen reduction chemistry and proton translocation, remains unknown. Crystal structures of various equilibrium forms of the enzyme have been used in the past to guide the formulation of proton translocation models, but so far, no consensus has been reached, as the structures were obtained with crystals prepared in the presence of a high level of cryoprotectants and with intense synchrotron radiation at cryogenic temperatures. It has been shown that under these conditions CcO is susceptible to dehydration and radiation-induced reduction, photodissiciation and protein damage. In addition, no structures of catalytic intermediates have been reported. This project is aimed at overcoming these obstacles by obtaining crystal structures of transient intermediates of bovine CcO (bCcO) under near- physiological conditions for the first time. This goal will be achieved by using Serial Femtosecond X-Ray Crystallography (SFX) with the X-Ray Free-Electron Laser (XFEL) at the Linac Coherent Light Source (LCLS) at SLAC, taking advantage of its unique ?diffraction before destruction? property. To achieve our goals two specific aims will be carried out. In Aim #1 we will use carbon monoxide (CO) as a surrogate for Oxygen (O2) and determine the structures of transient intermediates that are formed upon photodissociation of the CO from the enzyme and the structures that are formed upon reacting CO with the enzyme crystals. In Aim #2 we will determine the structures of the catalytic intermediates that are formed in the reaction of O2 with the enzyme. We have extensive preliminary data in which we have been able to solve structures of the reduced, oxidized and CO-bound species by SFX diffraction measurements and by mixing O2 with microcrystals we have obtained a structure of a transient species. The identity of the intermediates in all the crystals will confirmed by microscopic optical absorption and Raman spectroscopy that we have perfected in our lab. The expected outcome of this project is the determination of crystal structures of transient intermediates of bCcO at near- physiological conditions for the first time, thereby allowing the evaluation postulated proton translocation models and the formulation of new mechanisms.